Understanding how intratumor phenotypic heterogeneity promotes disease progression is essential to improve patient care. In this proposal we focus on the cooperative relationships between distinct tumor subpopula- tions, which are a critical yet poorly understood property of heterogeneity within tumors. We have recently uncovered a new symbiotic relationship between tumors subpopulations that promotes a transition from be- nign to malignant growth by inducing the collective invasion of cohesive groups of cells. Through analysis of the intrinsic heterogeneity within cell communities, we discovered an epigenetically distinct subpopulation of breast cancer ?trailblazer? cells that has an enhanced ability to initiate collective invasion. Importantly, sibling ?opportunist? cells can invade through paths in the ECM created by a minority subpopulation of trailblazer cells. This democratization of invasive behavior through subpopulation cooperation eliminates a bottleneck in tumor evolution, thus unleashing the metastatic potential of a more diverse tumor cell population. We have begun uncovering components of a unique multi-gene regulatory program that is specifically required for trail- blazer cell induced collective invasion and found evidence that it is active in patients with shorter survival times. Thus, we have revealed that the activation of a new signaling network in a subpopulation of cells can induce the formation of a novel cooperative relationship that yields widespread collective invasion and has the potential to negatively impact patient survival. Defining factors that control this new ?trailblazer? regulatory program and determining precisely how the interaction between trailblazer and opportunist cells contributes to cancer progression is necessary to explain how cooperative invasive behavior influences patient prognosis and reveal treatment options. Our overall objective in this proposal is to define how trailblazer and opportunist subpopulations influence tumor development. Our central hypothesis is that slow-cycling trailblazer cells in- duce metastasis by promoting the opportunistic invasion of a distinct subpopulation of metastasis initiating cells that lacks autonomous invasive ability. We will test our hypothesis and accomplish our objectives by: (1) defining factors that control the conversion between opportunist and trailblazer states; (2) determining how induction of the trailblazer state influences cell autonomous fitness and (3) determining how the cooperative relationship established between trailblazer and opportunist subpopulations contributes to metastasis. From our investigation, we expect to determine a new way in which heterogeneity promotes tumor development by revealing how the cancer hallmarks of proliferation and autonomous invasion can be distributed across distinct populations and shared in a synergistic relationship that promotes disease progression. These findings will support the development of a new mode of prognostic analysis directed towards identifying the presence and close spatial proximity of unique tumor cell subtypes. Deconstruction of processes that confer invasive ability through intercellular interactions may also uncover novel ways to thwart invasion.